Procedures for the reconstruction, primary culture and experimental use of rainbow trout gill epithelia

Abstract

This protocol describes how to reconstruct and culture the freshwater rainbow trout gill epithelium on flat permeable membrane supports within cell culture inserts. The protocol describes gill cell isolation, cultured gill epithelium formation, maintenance, monitoring and preparation for use in experimental procedures. To produce a heterogeneous gill epithelium, as seen in vivo, seeding of isolated gill cells twice over a 2-d period is required. As a consequence, this is termed the double-seeded insert technique. Approximately 5–12 d after cell isolation and seeding, preparations develop electrically tight gill epithelia that can withstand freshwater on the apical cell surface. The system can be used to study freshwater gill physiology, and it is a humane alternative for toxicity testing, bioaccumulation studies and environmental water quality monitoring.

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Figure 1: Diagram of the primary cultured rainbow trout epithelium.
Figure 2: Summarized flow diagram of rainbow trout gill cell isolation and seeding procedures.
Figure 3: Dissection of the gills and isolation of the cells.
Figure 4: Images of the double-seeded insert epithelium.
Figure 5: Membrane development can be monitored by daily TER measurements.

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Acknowledgements

This work was funded via a grant supporting S.S. from the National Centre for the Replacement, Refinement & Reduction of Animals in Research no. 26675, awarded to C.H. and N.R.B. L.C.S. was funded by a studentship from the Biotechnology and Biological Sciences Research Council (BBSRC) co-funded Case Award (BB/J500483/1) supported by the AstraZeneca Global Environment research program to N.R.B. and C.H. C.M.W.'s research is funded by a Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery grant. S.P.K. is funded by an NSERC Discovery Grant. S.F.O. is an employee of AstraZeneca. AstraZeneca is a biopharmaceutical company specializing in the discovery, development, manufacturing and marketing of prescription medicines. Funding bodies had no role in the design of the study or decision to publish. The work aims to identify effective alternatives to reduce, refine and replace the use of live animals to meet environmental regulatory testing needs.

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Contributions

S.S. and L.C.S. contributed equally to the manuscript and generated the data. S.S. developed the double-seeded inverted insert (DSII) technique. C.M.W., S.P.K. and P.P. developed the initial methodology. S.F.O., C.H. and N.R.B. supported the recent developments of the methods. C.H. and N.R.B. received funding for S.S. and L.C.S. that has enabled the development of the methods and expanded the use of the double-seeded insert (DSI) for the replacement of animals in toxicity testing and environmental monitoring. All authors contributed to the manuscript.

Corresponding authors

Correspondence to Sabine Schnell or Christer Hogstrand or Nic R Bury.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Preliminary results of the characterisation of the double seeded inverted insert (DSII)

(a) Measurements of TER in developing double-seeded inverted insert (DSII) epithelia from 3 days. Freshwater (blue points) was added (after the dotted line) to the companion well compartment (the apical cell surface, whilst the insert contained L-15 medium) on day 7 resulting in an increase in TER still evident 24 hours later on day 8 (n = 5). Values are means ± SEM. (b) The primary cultured rainbow trout gill epithelium is functionally polarised. Once the DSII epithelium has developed its signature resistance and is electrically tight (by day 10 in this case), and replacing the cell culture medium on the basolateral side (the companion well compartment) to fresh water (indicated by dotted line) causes a rapid reduction in TER (n = 3). Values are means ± SEM. (c) A scanning electron micrograph of the apical cell surface of DSII showing the irregularly shaped epithelial cells with microridges (mr) and plasma membrane (pm). Bar = 20 μm. (d) and (e) Transmission electron micrographs of DSII showing the tight junctions between epithelial cells (arrows) and the apical (AP) cell surface with glycocalyx and the basolateral cell surface (BL)) and the mitochondria rich cell (mitochondria = mc), the nucleus (n) and the intercellular space (S). In (d) bar = 200 nm and in (e) bar = 500 nm. (f) and (g) Confocal microscope images of DSI epithelia where 24 hrs before cell fixing, epithelia were exposed to (f) symmetrical conditions with L-15 medium on both sides of the epithelium or (g) asymmetrical conditions with FW in the upper compartment and L-15 medium in the companion well and cell nuclei were stained with 5 μM Hoechst (blue) and tight junctions with zonula occludens 1 antibody (green). Bar = 50 μm.

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Schnell, S., Stott, L., Hogstrand, C. et al. Procedures for the reconstruction, primary culture and experimental use of rainbow trout gill epithelia. Nat Protoc 11, 490–498 (2016). https://doi.org/10.1038/nprot.2016.029

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